^ACentre for Plant Conservation Genetics, Southern Cross University, Lismore NSW 2480, Australia
^BDepartment of Plant Science, Waite Campus, University of Adelaide, SA 5064, Australia

Introduction

The most widely used molecular marker system is based on the use of RFLPs. These are highly reliable and informative but they are technically demanding to assay and expensive. RFLPs still form the basis of many linkage maps and have proved to be valuable in monitoring specific traits. However, due to their many limitations more user-friendly markers are increasingly replacing them.

Marker assays based on the polymerase chain reaction (PCR) offer a far more user-friendly marker system. PCR based techniques such as RAPDs are unreliable and generally not transferable between crosses. However, microsatellites (di- or tri-nucleotide repeat sequences) also known as simple sequence repeats (SSRs) are highly reliable, co-dominant markers. Microsatellites are becoming more widely used for marker assisted breeding and variety identification due to their high level of polymorphism and ease of use.

SSRs have recently become important genetic markers in a wide range of crop species, including barley and wheat. They appear to be ubiquitous in higher organisms, although the frequency of microsatellites varies between species. They are abundant, dispersed throughout the genome, and show higher levels of polymorphism than other genetic markers. These features, coupled with their ease of detection have made them useful molecular markers. Their potential for automation and their inheritance in a codominant manner are additional advantages when compared to other types of molecular markers.

Variations in the length of tandem repeats can be identified by amplification of the region containing the repeat using PCR with primers designed to the regions flanking the SSRs. Polymorphisms are detected based on size differences which result from differences in the number of repeats. SSR loci are believed to evolve in a step-wise manner by the addition or subtraction of a single repeat. Up to 37 different alleles for the one SSR locus have been found in barley (Saghai Maroof et al. 1994).

Allele Typing of Barley Varieties Using SSRs

Forty-three barley microsatellite markers derived from (Liu et al. 1996) were used to genotype 180 barley varieties. Primer pairs were synthesised and screened on a panel of four genetically diverse genotypes. Seventeen of them giving unambiguous and polymorphic DNA banding patterns were selected for an allele typing study. Allele typing was carried out by using primers labelled with fluorescent dyes and the PCR products were separated and analysed on an ABI Prism 310 automated capillary electrophoresis system. Among 17 labelled primer pairs examined, four gave inconsistent PCR product results and were excluded from the study. The remaining 13 primer pairs detected a total of 135 alleles. Microsatellites with (GA)_n repeats revealed the highest number of different alleles.

A summary of results is shown below.

Variety Identification

Over 90% of the tested varieties could be uniquely identified by comparison of microsatellite allele sizes. A large number of additional SSR sequences have been made available to us by the Scottish Crop Research Institute (SCRI). These SSR sequences are being tested in our laboratory and mapped on double haploid populations developed by the National Barley Molecular Marker Program. Use of these SSRs should also enable us to develop tests to discriminate between all commercially grown barley varieties in Australia.

References

1. Liu, Z.-W., R.M. Biyashev and M.A. Saghai Maroof (1996). Development of simple sequence repeat DNA markers and their integration into a barley linkage map. Theoretical and Applied Genetics 93: 869-876.

2. Saghai Maroof, M.A., R.M. Biyashev, G.P. Yang, Q. Zhang and R.W. Allard (1994). Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations, and population dynamics. Proc Natl Acad Sci U S A 91: 5466-70.